Drugs for the diagnosis of tissue-reproductive activity or the treatment of proliferative diseases

ABSTRACT

An agent which comprises, as an active ingredient, a radiolabeled compound as represented by the following formula or a pharmaceutically acceptable salt thereof: 
                         
wherein R 1  denotes hydrogen, or a linear- or branched-chain alkyl group having 1–8 carbon atoms, R 2  denotes hydrogen, hydroxyl or a halogen substituent, R 3  denotes hydrogen or fluorine substituent, R 4  denotes oxygen, sulfur, or a methylene substituent, and R 5  denotes a radioactive halogen substituent.
 
     The agent is stable in vivo, and either stays in cells or is incorporated in DNA, thus serving for diagnosis of tissue proliferation activity or treatment of proliferative disease.

CROSS-REFERENCED APPLICATIONS

This application is a National phase of International ApplicationPCT/JP02/00408, filed 22 Jan. 2002, which designated the U.S. and thatInternational Application was not published under PCT Article 21(2) inEnglish.

TECHNICAL FIELD

The present invention relates to use of radiolabeled nucleosidederivatives for diagnosis of tissue proliferation activity and treatmentof proliferative diseases.

BACKGROUND ART

If proliferation activity of tumor cells can be determinednon-invasively by image diagnosis, it will be help for evaluation ofgrowth rate and malignancy of the tumor. Detection of the most rapidlygrowing regions of a tumor by image diagnosis will be useful inpreparing plans for radiation fields in radiotherapy and identifyingsuitable portions for biopsy. Such methods will permit an early andaccurate evaluation of therapeutic effects, which is difficult toidentify by CT- or MRI-based anatomical evaluation or PET-basedmeasurement of glucose-metabolic changes. Particularly, they will beuseful for an early assessment of therapeutic effects of anticanceragents that may cause strong side effects.

In order to solve these clinically important problems, use of5-iodo-deoxyuridine labeled with a radioactive iodine and thymidinelabeled with carbon-11 which is a positron-emitter, have been studied(Tjuvajev J G et al., J. Nucl. Med. 35, pp. 1407–1417 (1994); Blasberg RG et al, Cancer Res. 60, pp. 624–635 (2000); Martiat P h et al., J.Nucl. Med. 29, pp. 1633–1637 (1998); Eary J F et al., Cancer Res. 59,pp. 615–621 (1999); U.S. Pat. No. 5,094,835; U.S. Pat. No. 5,308,605).It is considered that these radiolabeled compounds are taken into cellsas precursors for DNA synthesis required for cell division ofrapidly-growing tumors, and then phosphorylated by thymidine kinase,followed by incorporation into DNA, to reflect proliferation activity ofthe tumor. These radiolabeled compounds, however, are decomposed rapidlyin vivo, making it difficult to perform non-invasive evaluation of theproliferation activity of the tumor. The method using carbon-11-labeledthymidine, in particular, requires very complicated mathematical modelanalysis, and cannot become popular as a diagnostic technique of nuclearmedicine imaging.

The rapid metabolic decomposition of these radiolabeled compounds invivo is considered to be due to cleavage of C—N glycosidic bonds bythymidine phosphorylase and instability of the labels in vivo. If theC—N glycosidic bonds are cleaved, the compound loses its affinity totumors, thereby decreasing in accumulation of radioactivity in tumors,while the radioactive metabolites increase background radioactivity,thereby making imaging of the tumors difficult.

To solve these problems, radiolabeled compounds with metabolic stabilityhave been synthesized by introducing fluorine atoms, which are high inelectronegativity, to the 2′ or 3′ position in certain nucleosides, andhave been studied for imaging of tumors. Thus,3′-deoxy-3′-fluorothymidine that contains fluorine 18, a positronemitter, at the 3′ position shows a high stability in vivo and anaccumulation in tumor tissue (Shields A F et al., Nature Med. 4, pp.1334–1336 (1998)). Though this radiolabeled compound is stable in vivo,it is a radio-labeled compound with a short-life positron emitter, andtherefore a cyclotron is required in the hospital, limiting the usage ofthe compound. For this radiolabeled compound, the major processresponsible for its accumulation in cells is the phosphorylation causedby thymidine kinase that is an index of DNA synthesis, and thus it doesnot serve as an agent that essentially reflects DNA synthesis.

A derivative of 5-iododeoxyuridine, in which fluorine is introduced tothe 3′ position in the same manner as above to increase its stability invivo, has recently been reported. Though stable in vivo, however, thisradiolabeled compound was high in retention in blood and failed to showa significant accumulation in a tumor compared to 5-iododeoxyuridine(Choi S R et al., J. Nucl. Med. 41, p. 233 (2000)).

2′-fluoro-5-iodoarabinouridine, in which fluorine is introduced to the2′ position, shows a high stability in vivo, and has been used foridentification of introduction and expression in vivo of a vector forgene therapy, utilizing a phosphorylation reaction specific to thymidinekinase of human herpesvirus. It has also been applied to image diagnosisfor virus infection, based on the high specificity to the viralthymidine kinase (Tjuvajev J G et al., Cancer Res. 56, pp. 4087–95(1996); Tjuvajev J G et al., Cancer Res. 58, pp. 4333–4441 (1998); WiebeL I et al., Nucleosides Nucleotides 18, 1065–1076 (1999); Gambhir S S etal., Nucl. Med. Biol. 26, pp. 481–490 (1999); Haubner R et al., Eur. J.Nucl. Med. 27, pp. 283–291 (2000); Tjuvajev J G et al. Cancer Res. 59,5186–193 (1999); Bengel F M et al., Circulation 102, pp. 948–950(2000)).

In view of the above situation, the present invention aims to provide aradiolabeled compounds that are practically useful in clinical fields,stable in vivo, and able to retain in cells after being phosphorylatedby thymidine kinase of mammals, or reflect the DNA synthesis activityafter being incorporated in DNA, particularly those compounds which arelabeled with a single-photon emitter to achieve a wide spectrum of use,and also aims to provide methods for diagnosis of tissue proliferationactivity and for treatment of proliferative disease, utilizing agentsthat contain said radiolabeled compounds.

DISCLOSURE OF THE INVENTION

To achieve the above-mentioned objectives, the present inventors havesynthesized a variety of radiolabeled compounds and have intensivelystudied to see if they are useful for image evaluation of tissueproliferation activity. As a result, the inventors have found thatradiolabeled compounds as represented by the following formula can servefor diagnosis of tissue proliferation activity or treatment ofproliferative disease, and have completed the present invention.

Specifically, the present invention provides an agent for diagnosis oftissue proliferation activity or for treatment of proliferative disease,which comprises, as an active ingredient, a radiolabeled compound asrepresented by the following formula or a pharmaceutically acceptablesalt thereof:

wherein R₁ denotes hydrogen, or a linear- or branched-chain alkyl grouphaving 1–8 carbon atoms; R₂ denotes hydrogen, hydroxyl, or a halogensubstituent; R₃ denotes hydrogen or fluorine substituent, R₄ denotesoxygen, sulfur or a methylene substituent, and R₅ denotes a radioactivehalogen substituent, excluding the case where R₁, R₂ and R₃ arehydrogen, R₄ being oxygen, and R₅ being radioactive fluorine, bromine,iodine, or astatine; the case where R₁ and R₃ are hydrogen, R₂ beingfluorine, R₄ being oxygen, and R₅ being radioactive bromine or iodine;and the case where R₁ and R₂ are hydrogen, R₃ being fluorine, R₄ beingoxygen, and R₅ being radioactive bromine or iodine.

The radiolabeled compounds of the present invention are stable in vivo,and can retain in cells after being phosphorylated by mammalianthymidine kinase or reflect the DNA synthesis activity after beingincorporated in DNA. Therefore, they realize effective diagnosis oftissue proliferation activity and treatment of proliferative disease,and are particularly useful as diagnostic radioactive imaging agents fordiagnosis of tissue proliferation activity or as radioactive therapeuticagents for treatment of proliferative disease in accordance withinternal radiotherapy, local radiotherapy or the like.

Thus, according to another aspect of the present invention, there areprovided methods for diagnosis of tissue proliferation activity, whichcomprise administering an effective amount of a radiolabeled compound asrepresented by the above formula or a pharmaceutically acceptable saltthereof to a mammal, followed by imaging in vivo distribution thereof,and methods for treatment of proliferative disease, which comprisesadministering an effective amount of said radiolabeled compound or saltto a mammal. Herein, mammal includes human beings.

In the present invention, the radiolabeled compounds as represented bythe above formula include salts thereof, or may be in a form of ahydrate or solvate of these. Such salts include pharmaceuticallyacceptable salts, for example, one formed with a mineral acid such ashydrochloric acid and sulfuric acid or with an organic acid such asacetic acid. As such a hydrate or solvate, mention may be made of thepresent radiolabeled compounds or salts thereof to which water moleculesor solvent molecules are attached. Furthermore, the compounds of thepresent invention include their various isomers such as tautomers.

In the above formula, the linear- or branched-chain alkyl group having1–8 carbon atoms as represented by R₁ includes, for example, methylgroup, ethyl group, propyl group, t-butyl group, and n-hexyl group, ofwhich methyl group is preferable. The halogen-substituent as representedby R₂ preferably includes fluorine, chlorine, and bromine. R₄ ispreferably oxygen or sulfur, of which sulfur is particularly preferable.

The radioactive halogen-substituent as represented by R₅ in the aboveformula includes F-18, Cl-36, Br-75, Br-76, Br-77, Br-82, I-123, I-124,I-125, I-131, and At-211, of which F-18, Br-76, I-123, and I-124 arepreferable for diagnostic purposes while Br-77, I-125, I-131, and At-211are preferable for therapeutic purposes.

Preferred compounds as represented by the above formula include thosewherein R₁ is hydrogen or methyl, R₂ is hydrogen or ahalogen-substituent, R₃ is hydrogen, and R₄ is oxygen or sulfur,particularly preferably those wherein R₁, R₂ and R₃ are hydrogen, R₄ issulfur, R₅ is a radioactive halogen-substituent selected from F-18,I-123, I-125, and I-131.

Certain 4′-thio nucleic acid derivatives as represented by the aboveformula (where R₅ is a non-radioactive halogen-substituent) have beenreported to be resistant to bacterial thymidine phosphorylase as aresult of studies on antiviral agents (Dyson M R et al., J. Med. Chem.34, pp. 2782–2786 (1991); Rahim S G et al., J. Med. Chem. 39, pp.789–795 (1996)). It has also been known that certain 5-iodine- and5-methyl-4′-sulfur substitution products inhibit phosphorylation ofthymidine by human thymidine kinase (Strosselli S et al., Biochem J.334, pp. 15–22 (1998)). The chemical structures of these compounds withsulfur at the 4′ position and their use as an antiviral agent arealready known (International Publication WO9101326, InternationalPublication WO9104982, Japanese Patent Laid-Open No. HEI 10-087687), butneither the corresponding radiolabeled compounds nor their use as aradioactive diagnostic imaging agent or radioactive therapeutic agenthave been known.

The chemical structures of certain compounds with a substituent at the1′ position as represented by the above formula (where R₅ is anon-radioactive substituent) and production methods thereof have alreadybeen known (Japanese Patent Laid-Open No. HEI 07-109289). However,neither the corresponding radiolabeled compounds nor their use as aradioactive diagnostic imaging agent or radioactive therapeutic agenthave been known.

The compounds as represented by the above formula can be used forvarious diagnoses of tissue proliferation activity and treatment forproliferative diseases by virtue of their in vivo stability and theircapability for retention in cells or capability for being incorporatedin DNA.

Such diagnoses of tissue proliferation activity include, for example,diagnosis of hyperplasia, regeneration, transplantation or viralinfection accompanied by abnormal proliferation.

The diagnosis of hyperplasia accompanied by abnormal proliferationincludes, for example, diagnosis of hyperplastic inflammation, benigntumors, or malignant tumors. The diagnosis of the hyperplasticinflammation includes, for example, diagnoses concerning activity ofchronic rheumatoid arthritis and determination of therapeutic effects.The diagnosis of the benign tumors includes, for example, diagnosesconcerning localization, activity and determination of therapeuticeffects. The diagnosis of the malignant tumors includes, for example,diagnoses concerning localization, progress, malignancy anddetermination of therapeutic effects, of primary and metastaticmalignant tumors. Benign tumors include, for example, prostatichyperplasia, endometrium hyperplasia (cystic hyperplasia, adenomyosisuteri, hysteromyoma), ovarian tumor (cystadenoma), mammary gland(mastopathy, mammary gland fibroadenoma), pituitary adenoma,craniopharyngioma, thyroid adenoma, adrenocortical adenoma andpheochromocytoma. Malignant tumors include, for example, malignantlymphoma (Hodgkin's disease, non-Hodgkin lymphoma), pharyngeal cancer,lung cancer, esophagus cancer, gastric cancer, colon cancer, hepaticcancer, pancreatic cancer, nephric tumor (nephric cancer,nephroblastoma), bladder tumor, prostatic cancer, testicular tumor,uterine cancer, ovarian cancer, breast cancer, thyroid cancer,neuroblastoma, brain tumor (primary brain tumor, metastatic braintumor), rhabdomyosarcoma, bone tumor (osteosarcoma, metastatic bonetumor), Kaposi's sarcoma, and malignant melanoma.

The diagnosis of regeneration accompanied by abnormal proliferation isexemplified by diagnosis of function of physiological regeneration ofblood and diagnosis of pathological regeneration resulting frompathological loss of blood cells, such as evaluation of physiologicalhematopoietic functions of bone marrow during treatment with anti-cancerdrugs and diagnosis of pathological functions of the bone marrow inpatients suffering from hypoplastic anemia.

The diagnosis of transplantation accompanied by abnormal proliferationis exemplified by diagnosis of blood cancer patients undergoing bonemarrow transplantation or very high-dose chemotherapy using ananticancer agent, such as diagnosis of take or proliferation oftransplanted bone marrow cells in bone marrow transplantation.

The diagnosis of viral infection accompanied by abnormal proliferationincludes, for example diagnosis of virus-infected portions andproliferation thereof in infectious diseases caused by Type I or Type IIherpes simplex virus, varicella-zoster herpes virus, cytomegalovirus,Epstein-Barr virus, or human immunodeficiency virus, particularlyinfectious diseases of central nervous system (e.g., viral-infectiouscerebritis, meningitis, etc.) caused by Type I or Type II herpes simplexvirus or human immunodeficiency virus.

The treatment for proliferative diseases is exemplified by treatment ofmalignant tumors or viral infection accompanied by abnormalproliferation. Such malignant tumors include, for example, malignantlymphoma (Hodgkin's disease, non-Hodgkin lymphoma), pharyngeal cancer,lung cancer, liver cancer, bladder tumor, rectal cancer, prostaticcancer, uterine cancer, ovarian cancer, breast cancer, brain tumor(primary brain tumor, metastatic brain tumor), and malignant melanoma.Such a viral infection includes infectious diseases of central nervoussystem caused by Type I or Type II herpes simplex virus or humanimmunodeficiency virus, particularly viral encephalitis or meningitis.

Methods for labeling the compounds represented by the above formula atthe “5” position with a radioactive halogen may be known methods, suchas methods using isotope exchange reaction, and a method using a5-chloromercuri compound in which mercury is introduced into the “5”position of the compound or a 5-hydrogen compound in which there is nosubstitution at the “5” position of the compound. The method using the5-chloromercuri compound is already known as an iodo-labeling method forproducing 5-iodo-2′-deoxyuridine (U.S. Pat. No. 4,851,520;Baranowska-Kortylewicz J et al., Appl. Radiat. Isot. 39, p. 335 (1988)).This method is, however, disadvantageous for producing pharmaceuticalslabeled with a short half-time radioactive nuclide due to side reactions(formation of “5-chloro” compounds, demercurization reaction), a longreaction time (6 hours), and formation of inorganic mercury compounds.The method using a 5-hydrogen compound is already known as a method forproducing 5-iodo-2′-deoxyuridine from 2′-deoxyuridine (Knaus E E et al.,Appl. Radiat. Isot. 37, p. 901 (1986); Fin R D et al., J. Label. Comds.Radiopharm. 40, p. 103 (1997)). This method, however, requires heatingat 65–115° C., and therefore, it is not suitable for use with compoundsthat are easily decomposed under heating conditions and cannot be saidto be an ideal labeling method, considering the properties ofradioactive halogen atoms which preferably should not involve heatingoperations during the labeling reaction. Further, the radiolabelingmethod using isotope exchange reaction is also unsuitable for producingpharmaceuticals that must be maintained at a certain level of quality,because the method is not able to produce carrier-free labeled compoundsand is difficult to control variation of specific activity amongdifferent labeling runs.

Another useful method for labeling the compounds represented by theabove formula at the “5” position with a radioactive halogen is to allowa compound (5-trialkyltin compound), in which the pyrimidine base issubstituted by a trialkylstannyl group at the “5” position asrepresented by Formula 11 in FIG. 1, Formula 21 in FIG. 2, Formula 28 inFIG. 3, Formula 40 in FIG. 4, Formula 50 in FIG. 5 or Formula 58 in FIG.6, to react with 0.1N sodium hydroxide solution of a radioactive halogenin an appropriate solvent such as chloroform, so that thetrialkylstannyl group at the “5” position is converted into aradioactive halogen-substituent. This labeling method, which uses a5-trialkyltin compound, is preferable as it does not suffer suchproblems as with the above three labeling methods. Specifically, thismethod requires only a relatively short reaction time, and it does notproduce “5-chloro” compounds or need heating as the reaction readilyproceeds at room temperature. The resulting labeled compounds are freeof carriers, and if a lower specific activity is desired, a labeledcompound with a fixed specific activity can be readily prepared byadding a carrier. This method is also featured in that purificationafter the reaction is easy to operate. Specifically, 5-trialkyltincompounds are largely different from the corresponding radioactivehalogen-labeled compounds in terms of overall molecular polarity as theelectrical properties at the “5” position differ between them. Owing tothe difference in the molecular polarity, labeled compounds andunreacted precursors can be separated easily by using a commercialreverse-phase silica gel cartridge after the labeling reaction. Thispermits elimination of the need of troublesome high performance liquidchromatographic purification.

Thus, according to another aspect of the present invention, there isprovided a method for producing a radiolabeled compound as representedby the following formula:

wherein R₁ denotes hydrogen or a linear- or branched-chain alkyl groupshaving 1–8 carbon atoms, R₂ denotes hydrogen, hydroxyl or a halogensubstituent, R₃ denotes hydrogen or fluorine substituent, R₄ denotesoxygen, sulfur or a methylene substituent, and R₅ denotes a radioactivehalogen substitient;comprising reacting a nucleoside derivative as represented by thefollowing formula:

wherein R₁ denotes hydrogen or a linear- or branched-chain alkyl groupshaving 1–8 carbon atoms, R₂ denotes hydrogen, hydroxyl or a halogensubstituent, R₃ denotes hydrogen or fluorine substituent, R₄ denotesoxygen, sulfur or a methylene substituent, and R₅ denotes atrialkylstannyl group, with an alkaline solution of a radioactivehalogen in a solvent, whereby the trialkylstannyl group of R₅ isconverted into the radioactive halogen substituent.

The 5-trialkyltin compounds as represented by Formula 11 in FIG. 1,Formula 21 in FIG. 2, Formula 28 in FIG. 3, Formula 40 in FIG. 4,Formula 50 in FIG. 5, and Formula 58 in FIG. 6 are novel compounds whichare useful intermediates for producing the radiolabeled compounds of thepresent invention.

Thus, according to another aspect of the present invention, there isprovided a compound as represented by the following formula:

wherein R₁ denotes hydrogen or a linear- or branched-chain alkyl groupshaving 1–8 carbon atoms, R₂ denotes hydrogen, hydroxyl or a halogensubstituent, R₃ denotes hydrogen or fluorine substituent, R₄ denotesoxygen, sulfur or a methylene substituent, and R₅ denotes atrialkylstannyl group.

In the above formula, the linear- or branched-chain alkyl groups having1–8 carbon atoms as represented by R₁ include, for example, methylgroup, ethyl group, propyl group, t-butyl group, and n-hexyl group, ofwhich methyl group is preferred. The halogen-substituent as representedby R₂ preferably includes fluorine, chlorine and bromine. R₄ ispreferably oxygen or sulfur. The trialkylstannyl group as represented byR₅ includes trimethylstannyl group, triethylstannyl group andtributylstannyl group.

Preferred compounds as represented by the above formula include thosewherein R₁ is hydrogen or methyl, R₂ is hydrogen or ahalogen-substituent, R₃ is hydrogen, and R₄ is oxygen or sulfur.

As seen from FIGS. 1–6, 5-trialkyltin compounds can generally besynthesized by providing their corresponding halogen-containing compound(as represented by Formula 10 in FIG. 1, Formula 20 in FIG. 2, Formula27 in FIG. 3, Formula 39 in FIG. 4, Formula 49 in FIG. 5, Formula 57 inFIG. 6) as starting materials, reacting the compound withbis(trialkyltin) and bis(triphenylphosphine)palladium chloride inanhydrous 1,4-dioxane under heat at reflux in an argon atmosphere,followed by purification.

Compound 10 (ITDU) in FIG. 1 can be synthesized by a known method(Formulae 1–8: Dyson, M R et al., Carbo. Res. 216, p. 237 (1991), andFormulae 8–10: Oivanen, M et al., J. Chem. Soc., Perkin Trans. 2, p.2343 (1998)). Specifically, 2-deoxy-D-erythro-pentose (Compound 1) isreacted with a 1% hydrochloric acid-methanol solution to produceCompound 2, which is then reacted with sodium hydride,tetrabutylammonium iodide, and benzyl bromide to produce Compound 3, inwhich hydroxyl groups are protected. The compound is reacted withα-toluenethiol and concentrated hydrochloric acid to produce Compound 4,which is then reacted with triphenylphosphine, benzoic acid, anddiethylazodicarboxylate to produce Compound 5. Sodium methoxide is thenused to remove the benzoyl group from Compound 5 to produce Compound 6,followed by its conversion into Compound 7 with methanesulfonylchloride. A ring is formed with sodium iodide and barium carbonate toproduce Compound 8, which is reacted with 5-iodouracil in the presenceof bistrimethylsilylacetamide and then with N-iodosuccinimide to produceCompound 9. Subsequently, Compound 9 is deprotected with titanicchloride to produce Compound 10.

Compound 20 (ITAU) in FIG. 2 can be synthesized by a known method(Formulae 13–17: Yoshimura Y et al., J. Org. Chem. 61, p. 822 (1996) andFormula 17–20: Yoshimura Y et al., J. Med. Chem. 40, p. 2177 (1997)).Specifically, 1,2;5,6-di-O-isopropylidene glucose (Compound 13) isreacted with sodium hydride and benzyl bromide to produce a 3-benzylcompound, which is subsequently reacted with hydrochloric acid, aqueoussodium periodate solution, and sodium borohydride to produce Compound14, which is then converted with hydrogen chloride into Compound 15. Thecompound is then reacted with mesyl chloride and sodium sulfide toproduce Compound 16, which is reacted with hydrochloric acid and sodiumborohydride successively to produce Compound 17. Hydroxyl groups areprotected with sodium hydride and benzyl bromide (Compound 18), and theresulting compound is converted to Compound 19 with m-chloroperbenzoicacid (m-CPBA) and acetic anhydride. It is further reacted with5-iodouracil in the presence of 1,1,1,3,3,3-hexamethylene disilazane(HMDS) to produce a glycosylated compound, which is then reacted withboron chloride to produce Compound 20.

Compound 27 in FIG. 3 can be produced as follows. Compound 17 shown inFIG. 2 is used as a starting material, which is reacted witht-butyldimethylsilyl chloride (TBDMSCl) in dimethylformamide (DMF) inthe presence of imidazole to protect the hydroxyl group at the “5”position with a silyl group to produce Compound 23.Trifluoromethanesulfonic acid anhydride (Tf₂O) is added thereto inpyridine to produce Compound 24 in which the hydroxyl group at the “2”position is trifluoromethanesulfonylated. The compound is reacted withpotassium fluoride, along with Kryptofix (registered trademark) 222 andpotassium carbonate, in acetonitrile, to produce a fluoride compound(Compound 25) in which the substituent at the “2” position isstereochemically reversed. The compound is reacted withm-chloroperbenzoic acid (m-CPBA) in methylene chloride and furthertreated with acetic anhydride to produce Compound 26. This is reactedwith the product resulting from a reaction of 5-iodouracil and1,1,1,3,3,3-hexamethylene disilazane (HMDS), and withtrifluoromethanesulfonic acid trimethylsilyl (TMSOTf). The resultingproduct is further treated with boron chloride in methylene chloride toproduce Compound 27.

Compound 39 (FIAU) in FIG. 4 can be synthesized by a known method(Formulae 30–37: Reichman U et al., Carbohydrate Res. 42, p. 233 (1975)and Formulae 37–39: Asakura J et al., J. Org. Chem. 55, p. 4928 (1990)).Specifically, Compound 31, which has been synthesized in four steps from1,2:5,6-di-O-isopropylidene glucose (Compound 30), is treated with acation exchange resin (Amberlite IR-120) to produce Compound 32, whichis then reacted with potassium periodate to produce Compound 33. This isthen reacted with sodium methoxide to produce Compound 34, followed byacetylation of hydroxyl groups to produce Compound 35. The compound istreated with a hydrogen bromide-acetic acid solution to produce Compound36, followed by condensation with an uracil derivative to produceCompound 37. It is subsequently reacted with diammonium cerium(III)sulfate (CAN) to produce Compound 38, followed by deprotection ofhydroxyl groups with sodium methoxide to produce Compound 39.

Compound 49 (FITAU) in FIG. 5 can be synthesized by a known method(Formulae 42–46: Yoshimura Y et al., J. Org. Chem. 62, p. 3140 (1997)and Formulae 46–49: Yoshimura Y et al., Bioorg. Med. Chem. 8, p. 1545(2000)). Specifically, Compound 43, which has been synthesized in ninesteps from 1,2:5,6-di-O-isopropylidene glucose (Compound 42), is reactedwith diethylaminosulfur trifluoride (DAST) to produce Compound 44, whichis then reacted with m-chloroperbenzoic acid (m-CPBA) to produceCompound 45. This is subsequently reacted with acetic anhydride toproduce Compound 46, which is reacted with trifluoromethanesulfonic acidtrimethylsilyl (TMSOTf) to cause condensation with a 5-iodouracilderivative to produce Compound 47. Finally, the two protective hydroxylgroups are removed to produce Compound 49.

Compound 57 (IMBAU) in FIG. 6 can be synthesized by a known method(Formulae 52–54: Itoh Y et al., J. Org. Chem. 60, p. 656 (1995) andFormulae 55–56: Asakura J et al., J. Org. Chem. 55, p. 4928 (1990)),combined with known reactions for protection and deprotection ofhydroxyl groups (Formulae 54–55 and Formula 56–57). Specifically,1-[3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-D-erythro-pento-1-enofuranosyl]uracil(Compound 52) is reacted with pivalic acid and bromosuccinimide (NBS) toproduce Compound 53, which is then reacted with trimethylaluminum toproduce Compound 54. The protection groups for hydroxyl groups areconverted from tert-butyldimethylsilyl to acetyl, followed by reactionwith diammonium cerium(III) sulfate (CAN) to produce Compound 56.Finally, the protection groups in Compound 56 are removed with ammoniato produce Compound 57.

For radiolabeled compounds of the present invention, appropriate dosesand routes of administration should be selected depending upon targetdiseases and objectives, but if they are used as an agent for diagnosisof tissue proliferation activity, a radioactivity in the range of 37 MBqto 740 MBq, preferably 111 MBq to 370 MBq is administered. Usually, theyare administered intravenously, but in some cases, other routes ofadministration including arterial or intraperitoneal administration anddirect administration to a tumor or other affected portions may be used.

If they are used as an agent for treatment of proliferative disease, aradioactivity in the range of 37 MBq to 7400 MBq, preferably 185 MBq to3700 MBq, is administered. Usually, they are administered intravenously,but in some cases, other routes of administration including arterial orintraperitoneal administration and direct administration to a tumor orother affected portions may be used. Furthermore, if they are used fortherapeutic purposes, the above dose may be administered several timesat appropriate intervals.

The agent for diagnosis of tissue proliferation activity of the presentinvention can serve for whole-body or local scintigraphy and whole-bodyor local SPECT imaging by use of nuclides for SPECT. Using nuclides forPET, they can also be applied for whole-body or local PET imaging.

The agent for diagnosis of tissue proliferation activity of the presentinvention can serve for quantitative determination of localproliferative activity based on appropriate model analysis. Furthermore,if non-proliferation tissue is used as a control, local proliferativeactivity can be defined easily in a semi-quantitative way.

The agent for treatment of proliferative disease of the presentinvention, when a beta-emitter such as I-131 is used therein, can servesto decrease large tumors of 1 cm or more in diameter, depending on therange of the ray. When an alpha-emitter such as At-211 is used, they canwork on small lesions of 0.1 mm or less in diameter more effectivelythan beta-emitter, and therefore, they are expected to serve fortreatment of micrometastasis over the body. Furthermore, nuclides thatemit Auger electrons, such as I-125, can have antitumor effects due toDNA breakage, only after labeled compounds have gathered around theDNAs. Therefore, suitable label nuclides for treatment of systemic tumorfoci including metastatic ones include alpha-emitter such as At-211, andbeta-emitter such as I-131 that can have effect on portions around thefoci depending on the range. The most effective method is the cocktailtherapy which uses a mixture of a compound labeled with an alpha-emitterand a compound labeled with a beta-emitter.

For treatment by local administration, compounds labeled with nuclidesthat emit Auger electrons, such as I-125, are particularly effective forbrain tumor that is difficult to remove completely by surgicaloperation, and residual tumor from malignant melanoma, and in view offunctional preservation, breast cancer, rectal cancer, prostatic cancer,and malignant mouth tumor, because they do no harm on portions otherthan pathologically proliferating cells owing to the properties of raysemitted therefrom. Technique for local administration includes, forexample, an administration into intracavitary foci such as colon cancerby use of an endoscope, a direct administration to foci affected bybrain tumor during craniotomy, and an administration by use of acatheter into an artery relevant to an affected organ such as liveraffected by cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a synthetic pathway for a compound of the presentinvention.

FIG. 2 illustrates another synthetic pathway for a compound of thepresent invention.

FIG. 3 illustrates a third synthetic pathway for a compound of thepresent invention.

FIG. 4 illustrates a fourth synthetic pathway for a compound of thepresent invention (5-trialkyltin compounds) and [I-125] FIAU producedtherefrom.

FIG. 5 illustrates a fifth synthetic pathway for a compound of thepresent invention.

FIG. 6 illustrates the other synthetic pathway for a compound of thepresent invention.

FIG. 7 illustrates a diagram showing in vivo label stability of [I-125]ITDU and [I-125] ITAU measured in Example 16, along with [I-125] IUR asa control.

FIG. 8 illustrates a diagram showing in vivo label stability of [I-125]ITDU, [I-125] ITAU, [I-125] FITAU and [I-125] IMBAU measured in Example17, along with [I-125] IUR (highly decomposable) as a control.

FIG. 9 illustrates a diagram showing in vivo distribution of [I-125]ITDU in normal mice measured in Example 18.

FIG. 10 illustrates a diagram showing in vivo accumulation of [I-125]ITDU, [I-125] ITAU, [I-125] FITAU and [I-125] IMBAU, along with [I-125]IUR as a control, in proliferating tissue measured in Example 18.

FIG. 11 illustrates a photograph (biological morphology) showing ascintigram of Walker tumor observed in Example 19.

EXAMPLES

The present invention will be described in detail below with referenceto examples, but is not limited to these examples.

Example 1

Synthesis of 5-trimethylstannyl-4′-thio-2′-deoxyuridine (Compound 11)

As shown in FIG. 1,benzyl-3,5-di-O-benzyl-2-deoxy-1,4-dithio-α,β-D-erythro-pentofuranoside(Compound 8) was synthesized, using 2-deoxy-D-erythro-pentose(Compound 1) as starting material, according to the method of Dyson M Ret al. (Carbo. Res. 216, p. 237 (1991)). Further,5-iodo-4′-thio-2′-deoxyuridine (ITDU: Compound 10) was produced fromCompound 8 according to the method of Oivanen M et al. (J. Chem. Soc.,Perkin Trans. 2, p. 2343 (1998)). Compound 10 was then used as astarting material to produce 5-trimethylstannyl-4′-thio-2′-deoxyuridine(Compound 11) according to the following procedure.

Compound 10 (9.5 mg, 0.026 mmol), bis(trimethyltin) (17.3 mg, 0.052mmol) and bis(triphenylphosphine)palladium(II) chloride (5 mg) weredissolved in anhydrous 1,4-dioxane (3 mL) under argon atmosphere, andafter heating at reflux for 3 hours, concentrated under reducedpressure. The residue was purified by silica gel thin layerchromatography (chloroform-methanol, 6:1) to produce the target Compound11 (6.9 mg, 65%).

1H NMR (270 MHz, CD₃OD) δ 0.26 (s, 9H, CH₃Sn), 2.26 (ddd, 1H, J=4.6,7.9, 13.2 Hz, 1H, H-2′), 2.27 (ddd, J=4.6, 6.6, 13.4 Hz, 1H, H-2′), 3.41(m, 1H, H-4′), 3.71 (dd, J=5.9, 11.2 Hz, 1H, H-5′), 3.80 (dd, J=4.6,11.2 Hz, 1H, H-5′), 4.47 (q, J=4.0 Hz, 1H, H-3′), 6.41 (t, J=7.2 Hz, 1H,H-1′), 7.93 (s, 1H, H-5).

Example 2

Synsthesis of [I-125]-5-iodo-4′-thio-2′-deoxyuridine ([I-125] ITDU:Compound 12)

To 0.1N sodium hydroxide solution (50 μL) of [I-125]-sodium iodide (33MBq), water (1 mL) and chloroform (1 mL) were added, and then chloroformsolution (4.7 μL) of iodine (60 μg, 0.47 μmol) was added, and shaken for10 seconds. After removing only the aqueous layer, ethyl acetatesolution (100 μL) of Compound 11 (100 μg, 0.25 μmol) was added, and theresulting solution was left to stand at room temperature for 2 hours.One drop of 1N sodium thiosulfate solution was added, and chloroform wasevaporated. After adding water (1 mL), the solution was passed through aSep-Pak Plus QMA cartridge column. The column was washed with water (0.5mL×2), and the resulting aqueous solution was combined to produceI-125-labeled Compound 12 (7.3 MBq, 22%).

Example 3

Synthesis of [I-123]-5-iodo-4′-thio-2′-deoxyuridine ([I-123] ITDU:Compound 12)

To 0.1% ammonium iodide solution (1 mL) containing [I-123]-ammoniumiodide (2.0 GBq), 1N hydrochloric acid (0.1 mL) and chloroform (1 mL)were added, and then chloroform solution (4.7 μL) of iodine (60 μg, 0.47μmol) was added, and shaken for 10 seconds. After removing only theaqueous layer, ethyl acetate solution (100 μL) of Compound 11 (100 μg,0.25 μmol) was added and left to stand at room temperature for 2 hours.One drop of 1N sodium thiosulfate solution was added, and chloroform wasevaporated. After adding water (1 mL), the solution was passed through aSep-Pak Plus QMA cartridge column. The column was washed with water (0.5mL×2), and the resulting aqueous solution was combined to produceI-125-labeled Compound 12 (228 MBq, 15%).

Example 4

Synthesis of 5-trimethylstannyl-1-(4-thio-D-arabinofuranosyl)uracil(Compound 21)

As shown in FIG. 2, 1,4-anhydro-3-O-benzyl-4-thio-α-D-arabitol (Compound17) was synthesized from 1,2;5,6-di-O-isopropylidene glucose (Compound13) according to the method of Yoshimura Y et al. (J. Org. Chem. 61, p.822 (1996)). Then, 5-iodo-1-(4-thio-D-arabinofuranosyl)uracil (ITAU:Compound 20) was produced from Compound 17 according to the method ofYoshimura Y et al. (J. Med. Chem. 40, p. 2177 (1997)). This Compound 20was used as a starting mateiral to produce5-trimethylstannyl-1-(4-thio-D-arabinofuranosyl)uracil (Compound 21) bythe following procedure.

Compound 20 (4.0 mg, 0.010 mmol), bis(trimethyltin) (6.6 mg, 0.020 mmol) and bis(triphenylphosphine)palladium(II) chloride (5 mg) weredissolved in anhydrous 1,4-dioxane (5 mL) in an argon atmosphere, andafter heating at reflux for 4 hours, concentrated under a reducedpressure. The residue was purified by silica gel thin layerchromatography (25% methanol/chloroform) to produce the target Compound21 (2.3 mg, 55%).

1H NMR (270 MHz, CD₃OD) δ 0.7 (s, 9H), 3.55–3.67 (m, 1H), 3.77–3.95 (m,2H), 4.07 (t, J=5.9 Hz, 1H), 4.16 (t, J=5.9, 1H), 6.28 (d, J=5.3 Hz,1H), 8.03 (s, 1H).

Example 5

Synthesis of [I-125]-5-iodo-1-(4-thio-D-arabinofuranosyl)uracil ([I-125]ITAU: Compound 22)

To 0.1N sodium hydroxide solution (50 μL) of [I-125]-sodium iodide (67MBq), water (1 mL) and chloroform (1 mL) were added, and then chloroformsolution (4.7 μL) of iodine (60 μg, 0.471 μmol) was added, and shakenfor 10 seconds. After removing only the aqueous layer, ethyl acetatesolution (100 μL) of Compound 21 (100 μg, 0.24 μmol) was added, and theresulting solution was left to stand at room temperature for 2 hours.One drop of 1N sodium thiosulfate solution was added, and chloroform wasevaporated. After adding water (1 mL), the solution was passed through aSep-Pak Plus QMA cartridge column. The column was washed with water (0.5mL×2), and the resulting aqueous solution was combined to produceI-125-labeled Compound 22 (17.3 MBq, 26%).

Example 6

Synthesis of5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-D-arabinopentofuranosyl)uracil(Compound 40)

As shown in FIG. 4,1-(3,5-di-O-acetyl-2-deoxy-2-fluoro-β-D-arabinopentofuranosyl)uracil(Compound 37) was synthesized from 1,2:5,6-di-O-isopylidene glucose(Compound 30) according to the method of Reichman U et al.(Carbohydrate, Res. 42, p. 233 (1975)). Further,5-iodo-1-(2-deoxy-2-fluoro-β-D-arabinopentofuranosyl)uracil (Compound39) was produced from Compound 37 according to the method of Asakura Jet al. (J. Org. Chem. 55, p. 4928 (1990)). This compound was used asstarting material to produce5-trimethylstannyl-1-(2-deoxy-2-fluoro-β-D-arabinopentofuranosyl)uracil(Compound 40) by the following procedure.

Compound 39 (5.0 mg, 0.013 mmol), bis(trimethyltin) (20.5 mg, 0.063mmol) and bis(triphenylphosphine)palladium(II) chloride (6.2 mg) weredissolved in anhydrous 1,4-dioxane (3 mL) in an argon atmosphere, andafter heating at reflux for 2 hours, concentrated under a reducedpressure. The residue was purified by silica gel thin layerchromatography (chloroform-methanol, 6:1) to produce the target Compound40 (3.6 mg, 66%).

1H-NMR(500 MHz, CD₃OD) δ 0.25 (S, 9H, CH₃Sn), 3.72 (dd, J=5.0, 12.0 Hz,1H, H-5′), 3.79–3.91 (m, H-4′), 4.33 (ddd, J=3.0, 5.0, 18.5 Hz, 1H,H-3′), 5.02 (td, J=4.0, 53.0 Hz, 1H, H-2′), 6.25 (dd, J=4.5, 16.0 Hz,1H, H-1′), 7.56 (S, 1H, H-5).

Example 7

Synthesis of[I-125]-5-iodo-1-(2-deoxy-2-fluoro-β-D-arabinopentofuranosyl)uracil([I-125] FIAU: Compound 41)

First, 0.1N sodium hydroxide solution of [I-125]-sodium iodide (80 MBq)was distilled off, followed by addition of methanol (1 mL), addition ofmethanol solution (4.8 μL) of iodine (61 μg, 0.48 μmol), and shaking for10 seconds. Then, methanol solution (100 μL) of Compound 40 (100 μg,0.24 μmol) was added, and the solution was left to stand at roomtemperature for 2 hours. One drop of 1N sodium thiosulfate solution wasadded, and methanol was evaporated. After adding water (1 mL), thesolution was passed through a Sep-Pak Plus QMA cartridge column. Thecolumn was washed with water (1.0 mL), and the resulting aqueoussolution was combined to obtain I-125-labeled Compound 41 (9.5 MBq,12%).

Example 8

Synthesis of5-trimethylstannyl-1-(2-deoxy-2-fluoro-4-thio-β-D-arabinopentofuranosyl)uracil(Compound 50)

As shown in FIG. 5,1-O-acetyl-3-O-benzyl-5-O-(tert-butyldiphenylsilyl)-2-deoxy-2-fluoro-4-thio-D-arabinopentofuranose(Compound 46) was synthesized from 1,2:5,6-di-O-isopylidene glucose(Compound 42) according to the method of Yoshimura Y et al. (J. Org.Chem. 62, p. 3140 (1997)). Further, Compound 46 was used to produce5-iodo-1-(2-deoxy-2-fluoro-4-thio-β-D-arabinopentofuranosyl)uracil(Compound 49) according to the method of Yoshimura Y et al. (Bioorg.Med. Chem. 8, p. 1545 (2000)). This compound was then used as a startingmateiral to produce5-trimethylstannyl-1-(2-deoxy-2-fluoro-4-thio-β-D-arabinopentofuranosyl)uracil(Compound 50) by the following procedure.

Compound 49 (5.0 mg, 0.013 mmol), bis(trimethyltin) (16.9 mg, 0.052mmol) and bis(triphenylphosphine)palladium(II) chloride (6.0 mg) weredissolved in anhydrous 1,4-dioxane (3 mL) in an argon atmosphere, andafter heating at reflux for 3.5 hours, concentrated under reducedpressure. The residue was purified by silica gel thin layerchromatography (chloroform-methanol, 6:1) to produce the target Compound50 (1.9 mg, 35%).

1H-NMR (500 MHz, CD₃OD) δ 0.26 (S, 9H, CH₃Sn), 3.61–3.68 (m, 1H, H-5′),3.80–3.81 (m, H-4′), 4.37 (td, J=6.0, 12.0 Hz, 1H, H-3′), 4.97 (td,J=5.5, 49.0 Hz, 1H, H-2′), 6.46 (dd, J=5.5, 11.5 Hz, 1H, H-1′), 7.99 (S,1H, H-5).

Example 9

Synthesis of[I-125]-5-iodo-1-(2-deoxy-2-fluoro-4-thio-β-D-arabinopentofuranosyl)uracil([I-125] FITAU: Compound 51)

First, 0.1N sodium hydroxide solution of [I-125]-sodium iodide (45 MBq)was distilled off, followed by addition of methanol (1 mL), addition ofmethanol solution (4.8 μL) of iodine (61 μg, 0.48 μmol), and shaking for10 seconds. Then, methanol solution (100 μL) of Compound 50 (100 μg,0.24 μmol) was added, and the resulting solution was left to stand atroom temperature for 2 hours. One drop of 1N sodium thiosulfate solutionwas added, and methanol was evaporated. After adding water (1 mL), thesolution was passed through a Sep-Pak Plus QMA cartridge column. Thecolumn was washed with water (1.0 mL), and the resulting aqueoussolution was combined to obtain I-125-labeled Compound 51 (3.5 MBq,7.8%).

Example 10

Synthesis of5-trimethylstannyl-1-methyl(2-deoxy-2-bromo-β-D-arabinopentofuranosyl)uracil([I-125] IMBAU: Compound 58)

As shown in FIG. 6,1-[2-bromo-3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-1-C-methyl-β-D-arabinofuranosyl]uracil(Compound 54) was produced from1-[3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-D-erythro-pento-1-enofuranosyl]uracil(Compound 52) according to the method of Itoh Y et al. (J. Org. Chem.60, p. 656 (1995)). Further,5-iodo-1methyl(2-deoxy-2-bromo-β-D-arabinopentofuranosyl)uracil(Compound 57) was produced from Compound 54 according to the method ofAsakura J et al. (J. Org. Chem. 55, p. 4928 (1990)). This compound wasused as starting material to produce5-trimethylstannyl-1-methyl(2-deoxy-2-bromo-β-D-arabinopentofuranosyl)uracil(Compound 58) by the following procdure.

Compound 57 (4.9 mg, 0.011 mmol), bis(trimethyltin) (16.0 mg, 0.049mmol) and bis(triphenylphosphine)palladium(II) chloride (5 mg) weredissolved in anhydrous 1,4-dioxane (3 mL) in an argon atmosphere, andafter heating at reflux for 2.5 hours, concentrated under a reducedpressure. The residue was purified by silica gel thin layerchromatography (chloroform-methanol, 6:1) to produce the target Compound58 (3.8 mg, 72%).

1H NMR (500 MHz, CD₃OD) δ 0.25 (S, 9H, CH₃Sn), 1.95 (S, 3H, 1′-CH₃),3.61–3.70 (m, 2H, H-5′), 4.08–4.11 (m, 1H, H-4′), 4.53 (d, J=3.0 Hz, 1H,H-5′), 4.79 (S, 1H, H-2′), 7.75 (S, 1H, H-5).

Example 11

Synthesis of[I-125]-5-iodo-1-methyl-(2-deoxy-2-bromo-β-D-arabinopentofuranosyl)uracil([I-125] IMBAU: Compound 59)

First, 0.1N sodium hydroxide solution of [I-125]-sodium iodide (62 MBq)was distilled off, followed by addition of methanol (1 mL), addition ofmethanol solution (4.0 μL) of iodine (51 μg, 0.40 μmol), and shaking for10 seconds. Then, methanol solution (100 μL) of Compound 58 (100 μg,0.20 μmol) was added, and the resulting solution was left to stand atroom temperature for 2 hours. One drop of 1N sodium thiosulfate solutionwas added, and methanol was evaporated. After adding water (1 mL), thesolution was passed through a Sep-Pak Plus QMA cartridge column. Thecolumn was washed with water (1.0 mL), and the resulting aqueoussolution was combined to obtain I-125-labeled Compound 59 (8.3 MBq,13%).

Example 12

Test for In Vitro Phosphorylation Activity of [I-125] ITDU and [I-125]ITAU

The phosphorylation activity of a labeled compound by thymidine kinasewas determined using a crude enzyme extracted from a mouse's lung cancercell strain LL/2. Liquid enzyme was extracted from a LL/2 mouse's lungcancer cell strain in the logarithmic growth phase according to themethod of Wolcott R M and Colacino J M (Anal. Biochem 178, p. 38–40(1989)). To a reaction liquid containing ATP, which is a phosphatedonor, 2 nmole of the label compound and the liquid enzyme were addedand reacted at 37° C. for a fixed period of time. The reaction wasstopped by adding 1 mL of a 100 mM lanthanum chloride/5 mMtriethanolamine solution. Phosphorylated material was preparated bycentrifugal separation to form a phosphate-metal complex, followed bymeasuring a radioactivity of the resulting precipitate with an automaticwell-type gamma counter (ARC-380, Aloka Co., Ltd.). Results are shown inTable 1 from which phosphorylation activity attributed to thymidinekinase was confirmed in both [I-125] ITDU and [I-125] ITAU.

TABLE 1 Phosphorylation activity of iodo-labeled nucleoside derivatives(n = 3) Iodo-labeled nucleoside Phosphorylated material production rateacids (p mole/mg protein/h) [I-125]ITDU 1182.7 ± 100.1 [I-125]ITAU 13.6± 6.6

Example 13

Test for In Vitro Metabolic Stability of [I-125] ITDU and [I-125] ITAU

To evaluate metabolic stability of glycosidic bond, decompositionreactivity for E. coli-originating thymidine phospholylase was studied.To the reacton liquid, 2 nmole of the labeled compound and 9 units of aliquid enzyme (Sigma Corporation) were added and reacted at 25□C for afixed period of time, and the reaction was stopped by treatment in aboiling water bath for 3 minutes. The reaction liquid was subjected tocentrifugal separation, and the supernatant was applied over a thinlayer silica gel plate along with an authentic standard (5-iodouracil:IU) and a non-labeled parent compound. It was developed with a mixtureof chloroform and isopropyl alcohol (3:1), and then autoradiography wasmeasured with a bioimaging analyzer (BAS-1500, Fuji Photo Film Co.,Ltd.). The area of interest was set to peak components of the Rf valuecorresponding to the authentic standard, and the amount of the resultingmetabolite was calculated from its proportion in percentage. Results areshown in Table 2 which indicates that [I-125] ITDU and [I-125] ITAU arestabler than 5-iododeoxyuridine ([I-125] IUR).

TABLE 2 C—N glycosidic bond cleavage activity for iodo-labelednucleoside derivatives (n = 3) Iodo-labeled 5-iodouracil productionrate* (relative nucleoside acids activity) [I-125]IUR 138606.2 ± 14902.3(1.00) [I-125]ITDU  4075.9 ± 736.4 (0.03) [I-125]ITAU   524.3 ± 373.8(<0.01) *p mole/units/30 min

Example 14

Evaluation of In Vitro Stability of Metabolism of VariousRadioactive-Iodine-Labeled Nucleic Acid Derivatives by ThymidinePhospholylase

To evaluate the metabolic stability of glycosidic bond in variousradioactive iodine-labeled nucleoside derivatives, their decompositionreactivity for E. coli-originating thymidine phospholylase was studied.To the reaction liquid, 0.5–12.0 nmol of the labeled compound and0.0009–9.0 units of a liquid enzyme (Sigma Corporation) was added andreacted at 25□C for a fixed period of time, followed by treatment in aboiling water bath for 3 minutes to stop the reaction. As [I-125] IBMAUwas unstable under heat treatment, the reaction liquid was cooled withice to stop the reaction. The reaction liquid was subjected tocentrifugal separation, and the supernatant was applied over a silicagel plate along with an authentic standard (5-iodouracil: IU) and anon-labeled parent compound. It was developed with a mixture ofchloroform and isopropyl alcohol (3:1), and then the autoradiogram wasmeasured with a bioimaging analyzer (BAS-1500, Fuji Photo Film Co.,Ltd.). The area of interest was set to peak components of the Rf valuecorresponding to the authentic standard, and the amount of the resultingmetabolite was calculated from its proportion in percentage. In the caseof [I-125] FITAU AND [I-125] IMBAU, a reversed phase silica gel platewas used, and after developed with a mixture of methanol and water(3:7), an autoradiogram was measured with a bioimaging analyzer(BAS-1500, Fuji Photo Film Co., Ltd.) similarly to the [I-125] IUR andothers. Result of analysis are shown in Table 3 which indicates that[I-125] FITAU and [I-125] IMBAU are still stabler than [I-125] IUR

TABLE 3 C—N glycosidic linkage cleavage activity of iodo-labelednucleoside acid derivatives by thymidine phospholylase for (n = 3)Iodo-labeled nucle- 5-IU production rate Relative oside (pmol/units/0.5h) activity [I-125]IUR 138606.2 ± 14902.3 1.00 [I-125]ITdU 3778.7 ±692.0 0.03 [I-125]ITAU  514.8 ± 367.0 <0.01 [I-125]FITAU  0.5 ± 0.1<0.00001 [I-125]IMBAU 0.0 0.0

Example 15

Test of Thymidine Kinase-Dependent Incorporation into Celles UsingThymidine Kinase-Deficient Cells

Thymidine kinase-dependent incorporation of labeled compounds into cellswas studied based on difference in incorporation between thymidinekinase-deficient cell strains L-M (TK-) and their parent L-M cells. L-Mand L-M (TK-) cells in the logarithmic growth phase were planted on24-well plates, each carrying 2.0×10⁵ cells, and cultured overnight.Then 2 nmol of a radioactive iodine-labeled nucleoside derivative wasadded and allowed to be incorporated in the cells for one hour. Thecells were washed three times with an ice-cooled phosphate buffersolution, and dissolved in 0.1N NaOH, followed by determination ofdegree of radioactivity incorporated in the cells using an automaticwell-type gamma counter (ARC-380 or ARC-300, Aloka Co., Ltd.).Measurements were analyzed to make evaluations based on the amount ofthe incorporated label molecules per unit weight of cellular proteins.Results are shown in Table 4 which indicates that [I-125] ITdU and[I-125] FITAU were incorporated in cells in a thymidine kinase dependentway as in the case of the [I-125] IUR as a control.

TABLE 4 Incorporation of radioactive iodine-labeled nucleosides in L-Mand L-M (TK−) cells Incorporation Iodo-labeled (pmol/mg protein/h) (LM)/nucleosides L-M L-M (TK−) {L-M(TK−)} [I-125]IUR 77.80 ± 7.45  27.86 ±2.94  2.79* [I-125]ITdU 10.90 ± 1.48  3.94 ± 0.63 2.77* [I-125]ITAU 1.68± 0.28 1.20 ± 0.20 1.40** [I-125]FITAU 0.34 ± 0.05 0.21 ± 0.05 1.62****p < 0.0005, **p < 0.05, ***p < 0.01 (T-test)

Example 16

Test for In Vivo Label Stability of [I-125] ITDU and [I-125] ITAU

To evaluate in vivo label stability of [I-125] ITDU and [I-125] ITAU,tests were conducted to study the accumulation of free iodine in thethyroid gland in normal mice. A 370 KBq portion of each labeled compoundwas injected in each of 10 week old normal mice into its tail vein, andthree animals were sacrificed and anatomized at appropriate intervals.For a control, in vivo distribution of [I-125] IUR was also observed.Incorporation of radioactivity in the thyroid gland was measured with anautomatic well-type gamma counter (ARC-300, Aloka Co., Ltd.).Incorporated radioactivity in tissue was calculated as the administrateddose per gram of the tissue per unit time, and represented inpercentage, as shown in FIG. 7. Results indicate that the accumulatedradioactivity from [I-125] ITDU and [I-125] ITAU in the thyroid glandwas significantly smaller than that from the control [I-125] IUR,proving that the in vivo label stability of the agents is high.

Example 17

In Vivo Label Stability of Radioactive Iodine-Labeled NucleosidesDerivatives

To evaluate in vivo stability of deiodination against each radioactiveiodine-labeled nucleoside derivative, tests were conducted to studyaccumulation of free iodine in the thyroid gland of normal mice. A 185KBq of each labeled compound was injected in each of 10 week old normalmice (C57BL/6) into the tail vein, and three animals were sacrificed andanatomized at intervals longer than in Example 16. Incorporation ofradioactivity in the thyroid gland was measured with an automaticwell-type gamma counter (ARC-300, Aloka Co., Ltd.). Incorporatedradioactivity in tissue (% ID) was calculated as the administrated pergram of the tissue, and represented in percentage, as shown in FIG. 8.Results indicate that the accumulated radioactivity from [I-125] ITDU,[I-125] ITAU, [I-125] FITAU and [I-125] IMBAU in the thyroid gland wassignificantly smaller than that from the control [I-125] IUR (highlymetabolizable substance), proving that the in vivo label stability ofthe agents is high.

Example 18

In Vivo Distribution of [I-125] ITDU in Normal Mice

A 370 KBq of [I-125] ITDU was injected in each of 10 week old normalmice into the tail vein, and three animals were sacrificed andanatomized at appropriate intervals. Incorporation of radioactivity ineach tissue sample was measured with an automatic well-type gammacounter (ARC-300, Aloka Co., Ltd.). Incorporated radioactivity in tissuewas calculated as the administrated dose per gram of the tissue, andrepresented in percentage, as shown in FIG. 9. Results indicate that theaccumulated radioactivity in proliferating tissues, namely the thymusand the small intestine, was certainly higher than that innon-proliferating tissues, namely the brain, liver and muscle.

To evaluate the accumulation of each radioactive iodine-labelednucleoside derivative in proliferating tissues, tests were conducted tostudy in vivo distribution in normal mice. A 185 MBq of each labeledcompound was injected in each of 10 week old normal mice (C57BL/6)through its tail vein, and three animals were sacrificed and anatomizedat appropriate intervals. Incorporation of radioactivity in each tissuesample was measured with an automatic well-type gamma counter (ARC-300,Aloka Co. Ltd.). Incorporated radioactivity in tissue was calculated asthe administrated dose per unit weight of the tissue, and represented inpercentage (% ID/g). As shown in FIG. 10, results indicate that [I-125]IUR (positive control) and [I-125] ITDU have accumulated in largeamounts particularly in the thymus which is a proliferating tissue innormal young mice.

Example 19

Sintigraphy of Walker Tumor Using [I-123] ITDU

Malignant tumor, a typical proliferative disease, was observed byscintigraphy. Walker tumor cells were transplanted subcutaneously in theright inguinal region of Wistar rats. After the transplantation, 37 MBqof [I-123] ITDU was injected into the tail vein of rats that suffered apalpable tumor of about 20 mm that was suitable for scintigraphy. Eachtumor-transplanted rat was anesthetized with Ravonal four hours afterthe administration of a drug. Then the rat was fixed in the face-upposition and observed statically with gamma-camera imaging equipment(GCA-90B, Toshiba Corporation). Imaging was performed using ahigh-resolution medium-energy collimator to obtain images for 10 minuteswith a resolution of 256×256. Results are illustrated in FIG. 11 whichshows that [I-123] ITDU serves for clear imaging of transplanted tumors(indicated by an arrow) in Wister rats.

INDUSTRIAL APPLICABILITY

The radiolabeled compounds of the present invention are stable in vivo,and they either retain in cells after being phosphorylated by mammalthymidine kinase or are incorporated in DNA to reflect the DNA synthesisactivity, thus serving for diagnosis of tissue proliferation activityand treatment of proliferative diseases, particularly as radioactivediagnostic imaging agents for tissue proliferation activity diagnosisand as radioactive therapeutic agents for proliferative diseasetreatment by internal radiotherapy, local radiotherapy and the like.

1. An agent for diagnosis of tissue proliferation activity whichcomprises, as an active ingredient, a radiolabeled compound asrepresented by the following formula or a pharmaceutically acceptablesalt thereof:

wherein R₁ denotes hydrogen, or a linear- or branched-chain alkyl grouphaving 1–8 carbon atoms; R₂ denotes hydrogen, hydroxyl, or a halogensubstituent; R₃ denotes hydrogen or a fluorine substituent, R₄ denotes,sulfur, and R₅ denotes a radioactive halogen substituent.
 2. An agentfor diagnosis of tissue proliferation activity which comprises, as anactive ingredient, a radiolabeled compound as represented by thefollowing formula or a pharmaceutically acceptable salt thereof:

wherein R₁ denotes hydrogen, or a linear- or branched-chain alkyl grouphaving 1–8 carbon atoms; R₂ denotes hydrogen, hydroxyl, or a halogensubstituent; R₃ denotes hydrogen or a fluorine substituent, R₄ denotes,sulfur and R₅ denotes a radioactive halogen substituent selected fromthe group consisting of F-18, Cl-36, Br-75, Br-76, Br-77, Br-82, I-123,I-124, I-125, I-131 and At-211.
 3. An agent for diagnosis of tissueproliferation activity which comprises, as an active ingredient, aradiolabeled compound as represented by the following formula or apharmaceutically acceptable salt thereof:

wherein R₁ denotes hydrogen, or a linear- or branched-chain alkyl grouphaving 1–8 carbon atoms; R₂ denotes hydrogen, hydroxyl, or a halogensubstituent; R₃ denotes hydrogen or a fluorine substituent, R₄ issulfur, and R₅ denotes a radioactive halogen substituent.
 4. An agentfor diagnosis of tissue proliferation activity which comprises, as anactive ingredient, a radiolabeled compound as represented by thefollowing formula or a pharmaceutically acceptable salt thereof:

wherein R₁ is hydrogen or methyl group, R₂ is hydrogen or ahalogen-substituent, R₃ is hydrogen, R₄ is or sulfur, and R₅ denotes aradioactive halogen substituent.
 5. An agent for diagnosis of tissueproliferation activity which comprises, as an active ingredient, aradiolabeled compound as represented by the following formula or apharmaceutically acceptable salt thereof:

wherein R₁, R₂ and R₃ are each hydrogen, R₄ is sulfur, and R₅ is aradioactive substituent selected from the group consisting of F-18,I-123, I-125 and I-131.
 6. A method for producing a radiolabeledcompound as represented by the following formula:

wherein R₁ denotes hydrogen or a linear- or branched-chain alkyl groupshaving 1–8 carbon atoms, R₂ denotes hydrogen, hydroxyl or a halogensubstituent, R₃ denotes hydrogen or fluorine substituent, R₄ denotes,sulfur, and R₅ denotes a radioactive halogen substituent, said methodcomprising reacting a nueleoside derivative as represented by thefollowing formula:

 wherein R₁ denotes hydrogen or a linear- or branched-chain alkyl groupshaving 1–8 carbon atoms, R₂ denotes hydrogen, hydroxyl or a halogensubstituent, R₃ denotes hydrogen or fluorine substituent, R₄ denotes,sulfur, and R₅ denotes a trialkylstannyl group, with an alkalinesolution of a radioactive halogen in a solvent, whereby thetrialkylstannyl group of R₅ is replaced with a radioactive halogensubstituent.